European Journal of Pharmacology 765 (2015) 291–299

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Molecular and cellular pharmacology

Subcellular localization and internalization of the vasopressin V1B receptor Aki Kashiwazaki a, Yoko Fujiwara a, Hiroyoshi Tsuchiya a, Nobuya Sakai b, Katsushi Shibata b, Taka-aki Koshimizu a,n a b

Division of Molecular Pharmacology, Department of Pharmacology, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan Department of Functional Genomics, Graduate School of Pharmaceutical Sciences, Himeji Dokkyo University, Hyogo 670-8524, Japan

art ic l e i nf o

a b s t r a c t

Article history: Received 28 March 2015 Received in revised form 20 August 2015 Accepted 25 August 2015 Available online 28 August 2015

Only limited information is available on agonist-dependent changes in the subcellular localization of vasopressin V1B receptors. Our radioligand binding study of membrane preparations and intact cells revealed that a large fraction of the V1B receptor is located in the cytoplasm in unstimulated CHO cells, which is in contrast to the plasma membrane localization of the V1A and V2 receptors. Moreover, when the affinity of radiolabeled arginine-vasopressin ([3H]AVP) was compared between membrane preparations and intact cells, the affinity of [3H]AVP to the cell surface V1B receptors, but not the V1A receptors, was significantly reduced. Although the number and affinity of cell surface V1B receptors decreased, they became extensively internalized upon binding with [3H]AVP. Approximately 87% of cell surface-bound [3H]AVP was internalized and became resistant to acid wash during incubation with 1 nM [3H]AVP. By contrast, less ligand (35%) was internalized in the cells expressing the V1A receptor. Extensive internalization of the V1B receptors was partially attenuated by inhibitors of cytoskeletal proteins, siRNA against β-arrestin 2, or the removal of sodium chloride from the extracellular buffer, indicating that this internalization involves clathrin-coated pits. Together, these results indicate that the mechanism that regulates the number and affinity of V1B receptors in the plasma membrane is markedly distinct from the corresponding mechanisms for the V1A and V2 receptors and plays a critical role under stress conditions, when vasopressin release is augmented. & 2015 Elsevier B.V. All rights reserved.

Keywords: V1B vasopressin receptor Internalization Receptor trafficking Radioligand binding Acid wash Stable expression Chemical compounds studied in this article: Arginine vasopressin (PubChem CID: 644077)

1. Introduction The cellular localization of G-protein coupled receptors (GPCRs) is dynamically regulated by external stimuli and is a critical determinant of receptor function (Pierce and Lefkowitz, 2001). In addition to coupling with the relevant G-proteins, agonist-stimulated GPCRs are phosphorylated and undergo desensitization and internalization through sequential processes involving β-arrestin and clathrin-coated vesicles (Gainetdinov et al., 2004; von Zastrow and Williams, 2012). Understanding of these sequential events relies on the GPCRs that are located primarily in the plasma membrane under unstimulated conditions (Pierce and Lefkowitz, 2001). However, relatively little is known about the GPCR Abbreviations: AVP, arginine-vasopressin; C-terminus, carboxyl-terminus; CHO, Chinese hamster ovary; DAB, 3,3′-diaminobenzidine; DMEM, Dulbecco’s modified Eagle’s medium; GFP, green fluorescent protein; GPCRs, G-protein coupled receptors; HEK, human embryonic kidney; N-terminus, amino-terminus; PFA, paraformaldehyde n Corresponding author. Fax: þ 81 285 44 5541. E-mail address: [email protected] (T.-a. Koshimizu). http://dx.doi.org/10.1016/j.ejphar.2015.08.043 0014-2999/& 2015 Elsevier B.V. All rights reserved.

subpopulations that are initially distributed in the cytoplasmic compartments. Because many of the natural ligands for GPCRs are hydrophilic, receptor localization is critical for the ligands to access the receptors. Therefore, a full understanding of the cellular response to external stimuli requires the knowledge of how cytosolic GPCR subpopulations contribute to the dynamics of agonist-promoted receptor internalization and recycling (Luttrell and GestyPalmer, 2010). Among the three subtypes of vasopressin receptor (V1A, V1B and V2 receptors) (Koshimizu et al., 2012), V1A and V2 receptors are known to show different kinetics of internalization and recycling. After agonist stimulation, internalized V1A receptors rapidly recycle to the plasma membrane, whereas V2 receptors remain in the cytoplasm for a longer period (Innamorati et al., 1999; Oakley et al., 1999). By contrast, how the subcellular localization of V1B receptors is regulated after arginine-vasopressin (AVP) stimulation remains unclear. A previous study revealed that a short segment of the carboxyl-terminus (C-terminus) of the V1B receptor includes a signature amino acid sequence that targets the receptor protein for exit from the endoplasmic reticulum and sorting to the plasma membrane (Robert et al., 2005).

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The V1B receptor is most abundantly expressed in the anterior pituitary and, together with corticotropin-releasing hormone receptors, regulates adenocorticotrophic hormone secretion (Antoni, 1993; Lolait et al., 1995). Studies using pharmacological tools and genetically modified rodent models have shown that the V1B receptor is a critical regulator of responses to several stress conditions (Aguilera et al., 2008; Lolait et al., 2007a, 2007b; Tanoue et al., 2004). Therefore, changes in the subcellular localization of the V1B receptor may directly affect the stress response in the hypothalamo–pituitary–adrenal axis (Aguilera et al., 2008; Roper et al., 2011). Here, we report the subcellular localization of V1B receptors and dynamics of their internalization, which are fundamentally different from those previously reported for V1A and V2 receptors.

2. Materials and methods 2.1. Materials Construction of the vectors for the expression of mouse V1B receptors was performed as previously described (Kikuchi et al., 1999; Oshikawa et al., 2004). The cDNAs for the mouse V1A receptors were obtained from total brain RNA by RT-PCR, and the nucleotide sequence was confirmed as previously described (Koshimizu et al., 2010). The receptor cDNAs were transferred to the eukaryotic expression vector pcDNA3.1 (Life Technologies Japan, Tokyo, Japan). A FLAG tag was introduced into the aminoterminus (N-terminus) next to the first methionine of the V1A and V1B receptors (FLAG-V1A and FLAG-V1B, respectively) or into the C-terminus immediately before the stop codon of the V1A and V1B receptors (V1A-FLAG and V1B-FLAG, respectively). Green fluorescent protein (GFP) was fused to the C-termini of V1A and V1B by subcloning the receptor coding sequence without its stop codon into the EcoRI/BamHI site of the pEGFP vector (Takara Bio, Shiga, Japan). Chinese hamster ovary (CHO)-derived cells and human embryonic kidney (HEK) cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and the RIKEN BioResource Centre (Ibaraki, Japan), respectively. U73122, U0126, SP600125, and AG1478 were purchased from Cell Signaling Technology (Tokyo, Japan). Latrunculin and nocodazole were purchased from Sigma-Aldrich Japan (Tokyo, Japan). Essential and non-essential amino acid cocktails and other chemicals of reagent grade were purchased from Wako Pure Chemical Industries (Osaka, Japan). 2.2. Cell cultures CHO and HEK293 cells were cultured in Ham’s F12 medium and in Dulbecco’s modified Eagle’s medium (DMEM), respectively, at 37 °C in 5% CO2 in an air-ventilated humidified incubator. Both culture media were supplemented with heat-inactivated 10% foetal calf serum (Life Technologies Japan), 100 U/ml penicillin, and 100 μg/ml streptomycin. To establish CHO cell lines that stably express V1A or V1B receptors (CHO/V1A or CHO/V1B), 1  105 cells were seeded in a 100-mm tissue culture dish. One day after seeding the cells, the medium was supplemented with a mixture of 0.7 mL of serum-free F12 medium, 21 μL of FuGene 6 transfection reagent (Promega, Tokyo, Japan) and 7 μg of the recombinant expression plasmid FLAG-V1A or FLAG-V1B, and the cells were cultured for 24 h. Single colonies that were resistant to the treatment of 0.2 mg/ml zeocin (Life Technologies Japan) were isolated and maintained in the same selection medium. The expression of the transfected receptors was screened using intracellular Ca2 þ measurement (Koshimizu et al., 2010). HEK cells were transfected as described for CHO cells and were used to monitor the cellular

localization of transiently expressed receptors. Small interfering RNAs for β-arrestin 1 and 2 were obtained from Life Technologies and were introduced into the cells according to the manufacturer’s instructions. 2.3. Radioligand binding study Membrane samples from CHO/V1A and CHO/V1B cells were prepared as described previously (Shibata et al., 1995). For saturation binding studies, 700 mg of membrane samples was incubated with 50–16,000 pM [3H]AVP (61.2 Ci/mmol, PerkinElmer Japan, Yokohama, Japan) for 60 min at 25 °C in 400 μl of binding buffer B (50 mM Tris–HCl, pH ¼7.4, 10 mM MgCl2, and 0.3% bovine serum albumin). At the end of the incubation period, 1 ml of icecold buffer B was added, and samples were filtered through a glass fibre membrane (type GF/B, GE Healthcare UK Ltd., UK). After three washes with buffer B, the filters were soaked in 4 ml of liquid scintillation cocktail (Ultima Gold™, PerkinElmer), and the membrane-bound radioactivity was measured using a liquid scintillation counter (LSC-6500; Hitachi Aloka Medical, Tokyo, Japan). The non-specific radioligand binding was determined in the presence of 1 μM AVP. Radioligand binding in intact cells grown in 12- or 24-well dishes was measured as described previously (Arai et al., 2015). For the saturation binding studies, CHO/V1A cells were grown in 24-well dishes and CHO/V1B cells in 12-well dishes because of the low level of cell surface V1B receptors. On the day of the experiment, the cells were washed once with ice-cold Ham's F-12 medium and incubated for 2 h on ice in buffer C [Ham's F-12 medium containing 50 mM HEPES (pH ¼7.4), 0.3% bovine serum albumin] with 10–30,000 pM of [3H]AVP. The cells were then washed 3 times with buffer B. The radioactivity bound to the cell surface receptors was collected using 0.5 ml of ice-cold acid buffer (50 mM sodium acetate, 150 mM NaCl, pH ¼3) and measured after mixing with 4 ml of the scintillation cocktail. The specific binding and Kd values were calculated using the nonlinear curve-fitting computer programme Igor (WaveMetrics, Lake Oswego, OR, USA). There was no detectable specific binding of [3H]AVP to the wildtype CHO cells. For the receptor internalization experiment, cells in 12-well culture plates were treated with 1 nM [3H]AVP in buffer C or basic buffer [10 mM HEPES (pH ¼7.4), 135 mM NaCl, 5 mM KCl, 1.2 mM CaCl2, 1 mM MgCl2, 10 mM glucose] containing vehicle or inhibitor of cytoskeletal protein for 2 h at 4 °C. The culture plates were subsequently incubated for 30 min at 4 °C on ice in a refrigerator or at 37 °C in an air incubator. For incubation at 37 °C, the temperature inside the air incubator was equilibrated for at least 2 h. After the two types of incubation, all plates were washed three times with ice-cold neutral buffer at 4 °C to remove excess [3H] AVP. In this study, the internalized [3H]AVP and [3H]AVP bound to cell surface receptors were termed “internalized sites” and “surface sites”, respectively. To measure the internalized sites separately from the surface sites, the cells were incubated with ice-cold acid buffer for 5 min at 4 °C to remove [3H]AVP bound to cell surface receptors and washed three times with basic buffer (Haigler et al., 1980). 2.4. Immunohistochemistry A rat monoclonal antibody against mouse V1B receptors was generated using a synthetic peptide corresponding to the third intracellular loop of the V1B amino acid sequence from lysine-228 to serine-248 (CLEA Japan, Tokyo, Japan). For immunocytochemistry, cells grown on 35 mm glass base dishes (AGC Techno Glass, Japan) were treated with 4% paraformaldehyde (PFA) for 1 h at 37 °C, washed once with phosphate buffer (pH ¼7.4), and

To screen hybridoma clones producing anti-V1B monoclonal antibodies by Western blot analysis, FLAG-V1B receptors expressed in HEK cells were immunoprecipitated with anti-FLAG antibody or anti-V1B monoclonal antibodies, separated by gel electrophoresis, and electrically blotted onto polyvinylidene fluoride membrane as previously described (Koshimizu et al., 2002). After blocking the membrane with 3% bovine serum albumin in TBS (10 mM Tris– HCl, pH ¼7.5, and 150 mM NaCl), the blots were detected using anti-FLAG or anti-V1B monoclonal antibodies at a dilution of 1:2000 or 1:100, respectively. Secondary anti-mouse or anti-rat antibodies conjugated with horseradish peroxidase were used at a dilution of 1:5000, and the signals were visualized with enhanced chemiluminescence (Western Lightning Plus ECL, PerkinElmer). 2.6. Statistics All values in the text are reported as the mean 7S.E.M. Significant differences were determined by Student’s t-test or ANOVA followed by a multiple comparison test with Holm’s adjustment. The statistics were calculated using the statistical computer programme R [R Core Team (2014). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria].

V1A

600 400 200 0

Binding sites (fmol/mg protein)

2

4

6 8 10 12 [3H]AVP (nM)

14 16

800 600

V1B

400 200 0 0

Binding sites (fmol/105 cells)

2.5. Western blot analysis

293

800

0

2

4

6 8 10 12 [3H]AVP (nM)

14 16

500

V1A

400 300 200 100

V1B

0 0

Binding sites (fmol/105 cells)

treated with 0.25% Triton X-100 and 5% dimethyl sulfoxide in phosphate buffer for 5 min under ambient conditions. Then, after treatment with a blocking buffer (3% bovine serum albumin in phosphate buffer), the cells were incubated with primary antibodies at the following dilutions: anti-FLAG M2 antibody (SigmaAldrich Japan) at 1:1500 and rat monoclonal antibody against mouse V1B receptors at 1:100. Secondary antibodies labelled with Alexa 488 or 594 were used at a dilution of 1:1000. For immunohistochemical analysis of rat pituitaries, male Wister-Kyoto rats, 8 weeks of age (Charles River Laboratories Japan, Yokohama, Japan), were perfused with 4% PFA, and the pituitary was immersed in 20% sucrose at 4 °C for 24 h. Frozen sections 8 μm in thickness were placed on slides, washed three times with phosphate buffer and treated with 1% Triton X-100 in phosphate buffer for 3 min. The blocking and staining steps were performed according to the manufacturer’s protocol (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA, USA), and the signal was visualized using 3,3′-diaminobenzidine (DAB) and nickel chloride as substrates. Anti-rat V1B antibodies (MBL, Nagoya, Japan) were used at a dilution of 1:1000. All of the animal experiments described above were approved by the Animal Care and Use Committee of Jichi Medical University and conducted in accordance with our institutional standards of animal care.

Binding sites (fmol/mg protein)

A. Kashiwazaki et al. / European Journal of Pharmacology 765 (2015) 291–299

2

4 6 [3H]AVP (nM)

8

10

25 20

V1B

15 10 5 0 0

5

10 15 20 [3H]AVP (nM)

25

30

3.1. A large fraction of AVP biding sites is located intracellularly in CHO/V1B cells

Fig. 1. Saturation binding studies of CHO/V1A and CHO/V1B cells. (A) Saturation binding experiments were performed on membrane samples prepared from CHO/ V1A and CHO/V1B cells with increasing concentrations of [3H]AVP at 25 °C as described in Section 2. Data were fitted to a four-parameter logistic equation, and Bmax and Kd values were calculated. Data shown are representative of an experiment performed in duplicate. Similar results were obtained in three experiments, and the mean values were calculated (Table 1). (B) Saturation binding experiments were performed on intact CHO/V1A and CHO/V1B cells with increasing concentrations of [3H]AVP at 4 °C. Mean values of the specific counts were plotted from a representative experiment and fitted to a four-parameter logistic equation to calculate Bmax and Kd values. Similar results were obtained in three or four experiments performed in duplicate (Table 1). (C) Y axis in (B) was extended to clearly show saturable binding to the surface V1B receptors. Data shown in (C) are mean values and S.E.M. from four experiments performed in duplicate. Non-specific binding was defined by incubating samples with 1 μM AVP.

To understand the regulatory mechanism of V1B receptor localization in reference to the V1A receptors, we first established stable CHO cell lines that express V1B or V1A receptors. Clonal cell lines were selected to keep the difference in the expression levels between V1A and V1B receptors within two fold in the radioligand binding study on membrane samples (Fig. 1A and Table 1). In contrast to the results from binding studies using membrane samples, studies of the ligand binding sites on the cell surface showed large differences between CHO/V1A and CHO/V1B cells; the Bmax values from the intact cell binding study of CHO/V1A and

CHO/V1B cells were 446 716 and 49 710 fmol/105 cells, respectively (Fig. 1B and Table 1). The binding of [3H]AVP to cell surface V1B receptors was saturable, as shown in Fig. 1C. Therefore, a large fraction of the V1B receptors expressed in CHO cells are located intracellularly and are inaccessible from the extracellular space by a hydrophilic ligand, in contrast to the plasma membrane localization of the V1A receptor. Another characteristic feature of CHO/ V1B cells is an increase in the Kd value in the intact cell binding study, compared with the values obtained from membrane samples; the Kd values of [3H]AVP binding to the intact cells were

3. Results

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Table 1 Bmax and Kd values obtained from saturation binding study on membrane samples or intact cells. °C CHO/V1A Membrane homogenates CHO/V1B Membrane homogenates

Kd (nM)

(n)

25 855 7 78

0.9 70.1

(3)

25 6457 53

1.8 70.7 (3)

°C CHO/V1A Intact Intact CHO/V1B Intact Intact a b

cells cells cells cells

Bmax (fmol/mg)

Bmax (fmol/105 cells) Kd (nM)

4 446 7 16a 37 281 7 14a 4 497 10b 37 2177 4b

1.4 70.1 1.1 70.2 32 717 11 71

(3) (3) (4) (3)

V1A-GFP

V1B-GFP

V1B

vector V1B IP: FLAG + — — — + — — — anti-V1B 1 — + — — — + — — anti-V1B 2 — — + — — — + — anti-V1B 3 — — — + — — — +

vector

V1B

+———+——— —+———+—— ——+———+— ———+———+

P o0.05, Bmax of intact CHO/V1A cells at 4 °C vs. 37 °C. Po 0.05, Bmax of intact CHO/V1B cells at 4 °C vs. 37 °C.

46 kDa

46 kDa

V1B

approximately 18 and 1.6 times larger than the values obtained from the membrane samples of CHO/V1B and CHO/V1A cells, respectively (Table 1). WB: anti-FLAG

3.2. Cellular localization of V1B receptors To confirm the findings of the binding experiments, the subcellular localization of V1A and V1B receptors was directly visualized. When fluorescence signals from the GFP-fusion receptors were examined (Fig. 2A), V1A-GFP was found primarily in the plasma membrane, as reported previously (Fukunaga et al., 2006), whereas a significant amount of signal from V1B-GFP was found in the cytoplasmic space (Fig. 2A). The cellular localization of untagged V1B receptors was determined after producing monoclonal antibodies against the V1B receptor. The localization of the V1B receptor obtained using primary anti-V1B antibodies was similar to the results for V1B-GFP (Fig. 2B). The specificity of the antibody clones was confirmed by the immunoprecipitation of FLAG-V1B expressed in HEK cells, followed by detection of the receptor on the blotting membrane (Fig. 2C). Immunohistochemical detection of pituitary V1B receptors in a tissue section also suggested that the V1B receptor is located both in the plasma membrane and in the intracellular space (Fig. 2D). The contrasting cellular localizations of V1A and V1B receptors were found in HEK cells as well as CHO cells (Fig. 2E). Adding FLAG tags to the N- or C-terminus or GFP to the C-terminus of the V1B receptor did not change the cellular localization of the receptors (Fig. 2E). By monitoring the intracellular Ca2 þ concentration during receptor activation, the responsiveness to 1 μM AVP and the cell surface expression of functional receptor protein were confirmed both in CHO/V1A and CHO/V1B cells and in HEK cells transfected with V1A or V1B receptors (data not shown). Our results strongly indicate that receptor localization differs between the V1A and V1B receptors despite their high amino acid sequence similarity (Koshimizu et al., 2012). 3.3. Internalization of [3H]AVP in CHO/V1A and CHO/V1B cells We next examined whether the difference in intracellular localization between V1A and V1B receptors could have any influence on receptor internalization. Washing cells with acetic acid buffer is known to remove the radioligand bound to cell surface receptors, termed here “surface sites", whereas washing with neutral buffer retains the specific counts of surface sites and radioligand associated with internalized receptors, termed “internalized sites”, as shown in Fig. 3A (Haigler et al., 1980). Incubation of the cells with 1 nM [3H]AVP at 4 °C prevented the internalization of agonist-

V1B

WB: anti-V1B 3

V1A-GFP

FLAG-V1B

V1B-FLAG

V1B-GFP

Fig. 2. Under unstimulated conditions, V1B receptors are localized primarily in the cytoplasm of cultured cells and anterior pituitary cells. (A) GFP-tagged V1A and V1B receptors at the C-termini were expressed in CHO cells. Cellular localization of GFP fluorescence (green) was examined under a microscope with a CCD camera. (B) V1B receptors without a tag sequence were expressed in CHO cells and detected by rat monoclonal antibodies raised against peptide sequence lysine-228 to serine-248 of the mouse V1B receptor, followed by anti-rat IgG Alexa 594 (red). (C) Immunoprecipitation and Western blot analysis of the V1B receptor with monoclonal anti-V1B antibody clones. HEK cells were transfected with vector alone (vector) or FLAG-V1B receptor gene (V1B). Cells were lysed 24 h after transfection, and the FLAG-V1B receptor was immunoprecipitated by anti-FLAG or V1B antibodies. Receptors were detected by Western blot analysis with anti-FLAG antibody or anti-V1B antibody clone 3. Data shown are representative of at least two independent experiments. (D) V1B receptor in the rat anterior pituitary was detected using anti-V1B antibody and sequential treatment with biotin-labelled secondary antibody and avidin-horseradish peroxidase. Positive signal was visualized with DAB and nickel chloride substrates. (E) Receptors expressed in HEK cells were detected with GFP fluorescence or anti-FLAG antibodies followed by anti-mouse Alexa 488 secondary antibodies (green). These figures are representative of three or four independent experiments. Scale bars in (A), (B), (D) and (E) indicate 20 μm.

bound V1A and V1B receptors (Fig. 3B and C, grey bars at 4 °C). Under these conditions, the acid wash effectively removed 99% of the surface sites (data not shown). To examine the internalization of the receptors, the culture plates at 4 °C were transferred to an incubator at 37 °C for 30 min (Fig. 3A). After the acid wash, higher radioactivity of internalized sites was detected in CHO/V1A cells than in CHO/V1B cells (Fig. 3B and C, white bars of internalized

22.5

Surface

Internalized

Surface + Internalized

4°C

4°C

4°C

37°C

4°C

37°C

Measurements Wash

Lysis

Measurements Wash Acid Lysis Wash

Measurements Wash

Lysis

3

1nM [ H]AVP

CHO/V1A

*

* 90

Binding sites (fmol/10 5 cells)

Binding sites (fmol/10 5 cells)

300 250 200 150 100 50

75

37 °C

300 250 200 150 100 50 0 5

10 15 20 25 [3H]AVP (nM)

30

V1B receptors (data not shown).

45 30

3.4. Saturable accumulation of [3H]AVP in CHO/V1B cells

0 4 °C

V1A V1B

60

15

0

295

Fig. 4. Surface plus internalized [3H]AVP binding sites at 37 °C are saturable in CHO/V1A and CHO/V1B cells. Increasing concentrations of [3H]AVP were incubated with cells at 37 °C for 30 min. Cells were then washed three times with neutral binding buffer and collected by lysis, as shown in Fig. 3A “Surface plus internalized”. Cell-associated radioligand, surface plus internalized sites, was measured by mixing cell lysate with scintillation cocktail and using a scintillation counter. Non-specific binding was determined in the presence of 1 μM AVP. Data shown are from three experiments performed in duplicate.

CHO/V1B

4 °C

37 °C

Surface

Surface

Surface + Internalized

Surface + Internalized

Internalized

Internalized

Fig. 3. Extensive internalization of V1B receptor in CHO cells. (A) Schematic representation of binding experiments. CHO cells expressing V1A or V1B receptor in a 12-well dish were incubated with 1 nM [3H]AVP at 4 °C for 2 h. Culture dishes were subsequently incubated at 4 or 37 °C for 30 min. After incubation, cells were placed on ice and washed three times with ice-cold neutral buffer to remove excess ligands. When cells were kept at 4 °C during the entire time course, internalization was inhibited and radioactivity associated with the cells were entirely from the surface sites (surface). Internalized [3H]AVP binding sites, “internalized sites”, were determined based on acid wash-resistant radioactivity associated with the cells. “Surface plus internalized sites” were determined by lysing cells after neutral buffer wash and measuring radioactivity. Results obtained on the CHO/V1A (B) and CHO/ V1B (C) cells were presented. Non-specific binding was determined in the presence of 1 μM AVP. *Po 0.05, surface sites at 4 °C vs. surface plus internalized sites at 37 °C. AVOVA analysis determined that both temperature and washing buffer had a significant influence on binding results. Data are from six experiments performed in triplicate.

sites at 37 °C), possibly because of the greater number of cell surface receptors in CHO/V1A cells. When the amount of internalized sites was converted to a percentage relative to the amount of surface sites at 4 °C, a significantly larger percentage of the surface sites was internalized in CHO/V1B cells compared with CHO/V1A cells; 87% and 35% of surface sites were internalized during incubation at 37 °C in CHO/V1B and CHO/V1A cells, respectively (Fig. 3B and C; white bars relative to grey bars, P o0.05). Extensive internalization of the V1B receptor resulted in an increase in the surface plus internalized binding sites at 37 °C. After the 37 °C incubation of the CHO/V1B cells, the surface plus internalized sites were significantly increased by 43% from the surface sites initially detected at 4 °C (Fig. 3C, black vs. grey bars, * indicates P o0.05). By contrast, the surface plus internalized sites associated with the CHO/V1A cells were decreased by 27% after the 37 °C incubation (Fig. 3B, black vs. grey bars, * indicates P o0.05). Agonist stimulation was necessary for the internalization of both V1A and V1B receptors, as changing the temperature from 4 to 37 °C in the absence of AVP did not induce internalization of the V1A or

We further examined the consequences of [3H]AVP binding and internalization at 37 °C. In both CHO/V1A and CHO/V1B cells, the surface plus internalized [3H]AVP-binding sites reached saturation, as shown in Fig. 4. Interestingly, the calculated Bmax value at 37 °C was significantly larger than the value obtained for the surface sites at 4 °C in CHO/V1B cells (Table 1; Figs. 1B and 4 for saturation binding at 4 and 37 °C, respectively). By contrast, the Bmax value of CHO/V1A cells at 37 °C was significantly smaller than the value obtained at 4 °C (Table 1; Figs. 1B and 4 for saturation binding at 4 and 37 °C, respectively). These results are consistent with the result shown in Fig. 3 and indicate that extensive internalization of V1B receptors resulted in an increased acid-resistant fraction of [3H]AVP, leading to accumulation of the surface plus internalized [3H]AVP sites at physiological temperature. The accumulation of a large fraction of internalized [3H]AVP in CHO/V1B cells could theoretically be due to the slower of removal of bound ligand from the V1B receptor. However, when the time course of ligand dissociation was examined, the rate of decline of bound ligand was faster in CHO/V1B cells than in CHO/V1A cells (Fig. 5). The time course of dissociation of [3H]AVP from cell surface receptors was examined at 4 °C to prevent agonist internalization. Therefore, the difference in ligand dissociation rate cannot explain the accumulation of internalized [3H]AVP in CHO/ 100 Bound (%)

Time (h)

Surface plus internalized sites (fmol/105 cells)

A. Kashiwazaki et al. / European Journal of Pharmacology 765 (2015) 291–299

*

80 60 V1A V1B

40 20 0 0

50 100 Time (min)

150

Fig. 5. Dissociation time course of [3H]AVP bound to CHO/V1A or CHO/V1B cells. Cells on 12-well dishes were placed on ice at 4 °C and incubated with 1 nM [3H]AVP in the presence or absence of 1 μM AVP for 1.5 h. Cells were washed three times with ice-cold binding buffer, and incubation was continued at 4 °C in the binding buffer. After the indicated periods of time, cells were washed three times with neutral buffer and collected by lysing with 0.1 N NaOH for radioactivity measurements. *Po 0.05, V1A vs. V1B. ANOVA analysis indicated that both receptor type and time have a significant influence on the remaining radioactivity. Data are from three experiments performed in duplicate.

A. Kashiwazaki et al. / European Journal of Pharmacology 765 (2015) 291–299

37 °C

β-arrestin 2

kDa

β-arrestin 1

siRNA

*

iz al rn

V1B cells. 3.5. Inhibition of V1B internalization by inhibitors of cytoskeletal proteins or by knockdown of β-arrestin 2 We searched for a candidate molecule involved in the process of V1B receptor internalization and found that simultaneous treatment with inhibitors of two cytoskeletal proteins, latrunculin as an actin polymerization inhibitor and nocodazole as a microtubule inhibitor, effectively reduced the V1B-associated internalization of [3H]AVP (Fig. 6, Internalized). This combination of inhibitors has been previously reported to reduce the recycling kinetics of single vesicles containing agonist-stimulated μ-opioid receptors (Roman-Vendrell et al., 2012). Treatment with either of these inhibitors individually was not sufficient to reduce V1B receptor internalization. The internalized receptor fractions in the CHO/V1B cells were 907 3, 897 5, and 75 77% in control buffer, latrunculin alone, and nocodazole alone, respectively (n ¼6). We detected no effect on V1B receptor internalization when the cells were incubated with the following enzyme inhibitors: 10 μM U73122 (an inhibitor of phospholipase C), 10 μM U0126 (an inhibitor of MEK1), 10 μM SP600125 (an inhibitor of JNK), or 0.1 μM AG1478 (an inhibitor of tyrosine kinases) (data not shown). The concentrations of these inhibitors were selected to inhibit corresponding enzyme activities to less than half. As observed for many GPCRs, β-arrestin is a critical regulator of V1B receptor internalization (Fig. 7). Knockdown of β-arrestin 2, but not of β-arrestin 1 (data not shown), specifically reduced the amount of β-arrestin 2 but not of ERK1/2, which is located downstream of β-arrestin 2-mediated signalling (Fig. 7A). Knockdown of β-arrestin 2 effectively reduced V1B receptor internalization compared with control siRNA at 37 °C, but no change

ERK1/2

100 50 0

Surface 200 [ 3H]AVP (%)

Fig. 6. Inhibition of V1B receptor internalization by simultaneous treatment with inhibitors of cytoskeletal proteins. CHO/V1B cells were incubated with 1 nM [3H] AVP in the presence of 10 μM latrunculin, an inhibitor of actin polymerization, and 10 μM nocodazole, an inhibitor of microtubule function, for 2 h at 4 °C and then for 30 min at 4 °C (surface sites) or 37 °C. Cells incubated at 37 °C were subsequently washed with ice-cold neutral (Surface and internalized sites) or acid buffer (internalized sites), lysed with 0.1N NaOH and subjected radioactivity measurements. Results were normalized to surface sites at 4 °C. *P o 0.05 vs. control. Data are from three to seven experiments performed in duplicate. Non-specific binding was determined in the presence of 1 μM AVP.

[3H]AVP (%)

In

Control Latrunculin + Nocodazole

β-arrestin 2

No siRNA Control siRNA β-arrestin 2 siRNA

te

Su rfa

ce fa ized r Su +nal r te in

ed

58 46 46 ce

Bound [ 3H]AVP (%)

4 °C 180 160 140 120 100 80 60 40 20 0

Control

296

*

150 100 50 0

Surface + internalized

Internalized

Fig. 7. Inhibition of V1B receptor internalization by treatment with siRNA against βarrestin 2. Small interfering RNA-mediated knockdown of β-arrestin 2 reduced the amount of β-arrestin 2, but not β-arrestin 1 or ERK1/2, in the CHO/V1B cells (A). In control and β-arrestin 2 siRNA-treated cells, incubation with 1 nM [3H]AVP at 4 °C resulted in similar amounts of binding to cell surface receptors (B). The amounts of bound [3H]AVP were shown relative to the bound [3H]AVP in the cells without siRNA transfection. Internalization of the V1B receptors was significantly inhibited in cells treated with siRNA against β-arrestin 2 (C). *Po 0.05 vs. control siRNA. Data are from three independent experiments performed in duplicate. Non-specific binding was determined in the presence of 1 μM AVP.

was detected in surface sites at 4 °C (Fig. 7C and B). 3.6. Sodium chloride-dependent internalization of the V1B receptor We next searched for a buffer condition that could alter extensive internalization of the V1B receptor and found that sodium chloride is required in the external buffer to induce V1B receptor internalization. Ham’s F12 medium includes 131 mM NaCl. When sodium chloride was removed from the basic buffer containing 135 mM NaCl, the radioactivity of the internalized sites was significantly reduced even after incubation of the CHO/V1B cells at 37 °C (Fig. 8A and B). Because the surface plus internalized sites in CHO/V1B cells were maintained even without added NaCl in the

200 160 120 80 40

fer

l AA Plu -es s sen tial Bu AA ffer wit hou tN aC Ha l m’s F12 me diu m

0

90 60 30

* P -es lus sen tial Bu AA ffer wit hou tN Ha aC m’s l F12 me diu m

ntia sse se

sic Ba

Plu

non

fer

l AA

0

buf

Acid resistant fraction (% of surface plus internalized sites)

Plu

non

se

Ba

sse

sic

ntia

buf

Surface plus internalized sites (fmol/10 5 cells)

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Fig. 8. Sodium chloride is necessary for V1B receptor internalization. The extent of ligand internalization was examined in different buffer conditions in CHO/V1B cells at 37 °C. Cells in 12-well dishes were incubated with 1 nM [3H]AVP for 30 min and washed three times with each buffer. Surface sites plus internalized binding sites were determined after washing cells with neutral buffer (A). Internalized sites were separately measured after the acid wash (B). Non-specific binding was determined in the presence of 1 μM AVP. Data presented are from six experiments performed in duplicate. *Po 0.05 vs. all other conditions. Essential AA indicates essential amino acids.

binding buffer, it is concluded that V1B receptor internalization is dependent on the extracellular NaCl and that bound [3H]AVP remained at the cell surface in the NaCl minus buffer (Fig. 8).

4. Discussion Here, we report that the cellular localization of the V1B receptor and its regulation by agonist are unique among vasopressin receptor family members. In two cell lines with similar expression levels in membrane preparations, significantly more total binding sites were found at the cell surface in CHO/V1A cells than in CHO/ V1B cells. Consistent with the results of this binding experiment, a marked difference in subcellular localization was found between the V1A and V1B receptors in the unstimulated state. The V1B receptor was found primarily in the cytoplasm, whereas the V1A and V2 receptors have been previously located primarily in the plasma membrane (Innamorati et al., 2001). We confirmed the subcellular localization of the V1B receptor using multiple techniques. First, GFP was fused to the receptor C-terminus. Second, antibodies to the V1B -specific sequence were developed and used to detect receptor proteins expressed in culture cells and in the anterior pituitary. Finally, FLAG peptide tags were added to the N- or

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C-terminus. Our results consistently showed that the V1B receptor was primarily cytoplasmic. Although the fraction of the V1B receptor that was sorted to the plasma membrane was relatively small, cell surface V1B receptors were functional. Specific binding of [3H]AVP to the cell surface of CHO/V1B cells was detected, and the concentration of intracellular Ca2 þ ions increased upon stimulation (Thibonnier et al., 1998). After agonist stimulation, a higher percentage of the receptor-bound [3H]AVP was internalized into the CHO/V1B cells than into the CHO/V1A cells. The internalization of the V1B receptor is dependent on elements of the cellular and extracellular environment, such as cytoskeletal proteins, β-arrestin, and extracellular sodium chloride. These results are important for understanding the unique nature of V1B receptor functions. Some GPCRs are localized in the cytoplasm in an unstimulated state, with or without plasma membrane localization. For example, the majority of α1D adrenoceptors are known to be present in the cytoplasm, with only a small number located in the plasma membrane (Chalothorn et al., 2002). By contrast, α1B receptors are localized primarily in the plasma membrane. In the case of the α1D adrenoceptor, the extracellular N-terminus is responsible for its retention in the cytoplasm (Hague et al., 2004). When the N-terminus of the human V1B receptor gene was modified to include the secretion signal from insulin, the C-terminal hydrophobic sequence, F341N(X)2LL(X)3L350, was necessary for the receptor protein to exit from the endoplasmic reticulum and reach the plasma membrane (Robert et al., 2005). We found that the subcellular localization of the mouse V1B receptor was not influenced by the addition of an artificial FLAG tag at either the N- or C-terminus or by the fusion of a GFP protein at the C-terminus. Furthermore, the intracellular distribution of the V1B receptor was conserved among transfected CHO and HEK cells as well as anterior pituitary cells. In previous reports, the human V1B receptor was also detected in the cytoplasm (Murat et al., 2012). In the case of the V2 receptor, a point mutation introduced in the DRY motif resulted in a constitutively desensitized and internalized receptor, which was unresponsive to AVP and exhibited the disease phenotype of nephrogenic diabetes insipidus (Barak et al., 2001). We found that the binding affinity of radiolabeled AVP to the cell surface V1B receptor was lower than the affinity obtained using the membrane preparation (Fig. 1, Table 1). This result suggests that the cellular environment and molecules that interact with the V1B receptor, such as G proteins, play important roles in agonist affinity. One interesting and unexpected finding in this study is that AVP was extensively internalized in V1B-expressing cells so that the amount of surface [3H]AVP sites plus internalized [3H]AVP sites exceeded the amount of [3H]AVP initially bound to the cell surface (Figs. 1B, 3, and 4, and Table 1). It remains unclear at present how this extensive internalization of the V1B receptors is maintained. An intracellular pool of cell surface receptors was postulated for the thromboxane A2 receptor C-terminal splice isoform (Parent et al., 2001), a constitutively active mutant of the angiotensin II type 1A receptor (Miserey-Lenkei et al., 2002), RNA-editingmediated isoforms of the 5-hydroxytryptamine 2c receptor (Marion et al., 2004), a C-terminal truncation mutant of the μ-type opioid receptor (Segredo et al., 1997) and protease-activated receptor 1 (Shapiro and Coughlin, 1998). However, in the case of the cannabinoid CB1 receptor, constitutively internalized receptors are trafficked to the lysosomal compartment and do not contribute to cell surface repopulation (Grimsey et al., 2010; Rozenfeld and Devi, 2008). Further study is needed to clarify the contribution of intracellular V1B receptors to the extensive internalization of the cell surface receptors. Recent studies suggest that the internalized receptors in the endosome are able to transduce signals (Irannejad et al., 2013; Tsvetanova et al., 2015). Intracellular β-adrenoceptors couple with the cognate heterotrimeric G protein Gs, and

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cannabinoid CB1 receptors in the late endosome or lysosomal compartments associate with Gi protein and phosphorylated ERK (Irannejad et al., 2013; Rozenfeld and Devi, 2008). It is not known whether extensively internalized V1B receptors could evoke intracellular signals, but the V1B receptor serves as a suitable model for the further investigation of structural and functional relationships among receptor population with different intracellular localizations. We found that V1B receptor internalization was dependent on cytoskeletal proteins, β-arrestin 2, and extracellular sodium chloride. These requirements suggest that clathrin-coated pits are involved in V1B receptor internalization. Microtubules and actin filaments have been implicated in endocytosis and vesicular traffic (Apodaca, 2001). Indeed, the recycling of β2 adrenoceptors to the plasma membrane required linking of the receptor to the local actin cytoskeleton (Puthenveedu et al., 2010). In addition, an intact actin and microtubule cytoskeleton was needed for proper recycling of μ-opioid receptors back to the plasma membrane (Roman-Vendrell et al., 2012). How these cytoskeleton proteins are involved during V1B receptor internalization and recycling needs to be clarified. Additionally, hypotonic shock was reported to inhibit the formation of coated pits (Larkin et al., 1983). Based on the results of the current study, analysis of intracellular trafficking of the V1B receptor would require a method for pulse-chase labelling of cell surface receptors and sensitive detection of the labelled receptors in the cells. Pulse-chase labelling should distinguish newly internalized receptors from receptors that are constitutively located in the cytoplasm. In summary, we found that the cellular localizations of the V1B receptors in agonist-stimulated and unstimulated conditions were remarkably distinct from the corresponding localizations of the V1A or V2 receptors. The V1B receptors undergo extensive internalization upon agonist binding. These characteristics of V1B receptors are important for understanding cellular responsiveness under agonist-stimulated conditions and useful for the development of a potential therapeutic intervention focused on V1Bmediated signals in the pituitary and other organs.

Acknowledgements We wish to thank Ms. Yuki Oyama for her technical assistance. This work was supported in part by Grants-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan [Grant 24590327] and the Promotion and Mutual Aid Corporation for Private Schools of Japan [no grant number assigned].

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